13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (V_{max app}) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s⁻¹·mg protein⁻¹), with a K_m value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg²⁺, Ca²⁺, Cu²⁺ and Cd²⁺, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

α,β-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,β-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin–Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.

Arabidopsis ( Arabidopsis thaliana ) 12-oxophytodienoic acid reductase isoform 3 (OPR3) is involved in the synthesis of jasmonic acid by reducing the α,β-unsaturated double bond of the cyclopentenone moiety in 12-oxo-phytodienoic acid. Recent research revealed that jasmonic acid synthesis is not strictly dependent on the peroxisomal OPR3. In addition, OPR3 is able to reduce trinitrotoluene suggesting that the old yellow enzyme homologue OPR3 has additional functions. Here we demonstrate that OPR3 catalyzes the reduction of a wide spectrum of electrophilic species that share a reactivity towards the major redox buffers glutathione (GSH) and ascorbate (ASC). Furthermore, we demonstrate that OPDA reacts with ascorbate to form an ASC-OPDA adduct, but in addition OPR3 has the ability to regenerate ASC from monodehydroascorbate (MDHA). The presented data characterize OPR3 as a bifunctional enzyme with NADPH-dependent α,β-ketoalkene double bond reductase and monodehydroascorbate reductase activities (MDHAR). opr3 mutants exhibited a slightly less reduced ASC pool in leaves in line with the MDHAR activity of OPR3 in vitro . These functions link redox homeostasis as mediated by ASC and GSH with OPR3 activity and metabolism of reactive electrophilic species (RES).